The mechanisms of proanthocyanidins Inhibits PGE_2 synthesis in lipopolysaccharide-Induced RAW264.7 cells

目的:观察原花青素对脂多糖诱导rAW264.7细胞PgE2生成的影响及其作用机制。方法:放射免疫法(rIA)检测原花青素对脂多糖诱导的rAW264.7细胞PgE2生成的影响;lPS诱导rAW264.7细胞9H,再加不同浓度原花青素作用30MIn,放射免疫(rIA)法检测原花青素对COX-2酶活性的影响;半定量逆转录-聚合酶联反应(rT-PCr)法检测原花青素对COX-2 MrnA表达的影响;提取核蛋白,蛋白免疫印迹(WESTErn blOT)法检测原花青素对nf-κb/P65蛋白表达的影响;电泳迁移率变动分析(EMSA)法检测原花青素对nf-κb与dnA结合活性的影响。结果:0.8,4和20Mg·l-1原花青素抑制lPS诱导rAW264.7细胞PgE2生成;0.8,4和20Mg·l-1原花青素不影响lPS诱导rAW264.7细胞COX-2酶活性;0.8,4和20Mg·l-1原花青素下调lPS诱导rAW264.7细胞COX-2 MrnA表达;4,20Mg·l-1原花青素下调lPS诱导rAW264.7细胞nf-κb/P65蛋白表达;0.8,4和20Mg·l-1的原花青素可明显降低lPS诱导下rAW264.7细胞的nf-κb活化。结论:原花青素显著抑制lPS诱导rAW264.7细胞PgE2生成的作用与原花青素抑制COX-2 MrnA表达有关,此作用可能是通过抑制nf-κb/P65蛋白表达和抑制nf-κb的dnA结合活性来实现。Objective:To study the effect of proanthocyanidins on PGE2 synthesis in lipopolysaccharide-induced RAW264.7 cells and its mechanisms.Methods: The effect of proanthocyanidins on PGE2 synthesis in lipopolysaccharide-induced RAW264.7 cells was measured by radioimmunoassay(RIA);After being pretreated with different concentrations of proanthocyanidins for 30 min,and then LPS 1 mg·L-1 for 9 h,the effect of proanthocyanidins on the activity of COx-2 enzyme in RAW264.7 cells was analysed by RIA;the expressions of COx-2 mRNA by RT-PCR;the nuclear protein was isolated and the expressions of NF-κB protein by Western blot;and the DNA-binding activity of NF-κB was measured by electrophoretic mobility shift assay(EMSA).Results: PGE2 synthesis was inhibited by proanthocyanidins 0.8 mg·L-1、4 mg·L-1 and 20 mg·L-1 ;the activity of COx-2 enzyme was not inhibited by proanthocyanidins 0.8 mg·L-1、4 mg·L-1 and 20 mg·L-1 (P>0.05,vs LPS group);the expression of COx-2 mRNA was inhibited by proanthocyanidins 0.8 mg·L-1、4 mg·L-1 and 20 mg·L-1;the expression of NF-κB/p65 protein was inhibited by proanthocyanidins 4 mg·L-1 and 20 mg·L-1.The DNA-binding activity of NF-κB was reduced by proanthocyanidins 0.8 mg·L-1、4 mg·L-1 and 20 mg·L-1.Conclusion: The mechanisms of proanthocyanidins inhibited PGE2 synthesis in lipopolysaccharide-induced RAW264.7 cells is related to the inhibition of the expression of COx-2 mRNA ,which is inhibited possibly by suppressing expression of NF-κB/p65 protein and DNA-binding activity of NF-κB.广东省教育厅自然科学基金(Z03045)---

Development of Electrochemical Biosensor for Detection of PML/RARα Fusion Gene in Acute Promyelocytic Leukemia

针对急性早幼粒细胞白血病(APl)中PMl/rArα融合基因的碱基序列,设计了锁核酸(lnA)修饰的发夹结构捕获探针,结合信号探针构建新型的“三明治“电化学传感模式。信号探针末端修饰的生物素可与酶上的亲和素结合,通过检测酶催化H2O2氧化底物3,3',5,5'-四甲基联苯胺(TMb)产生的电化学信号,实现对靶序列的检测。该传感器可识别和定量检测PbS缓冲液中人工合成的PMl/rArα融合基因序列。结果表明,该传感器能很好地区分互补序列、单碱基及多碱基错配序列,杂交电流值与目标链浓度在1.0x10-11~1.6x10-10 MOl/l范围内呈较好的线性关系,检出限为1.0x10-13 MOl/l。同时,该新型传感器成功地用于无稀释人血清中PMl/rArα融合基因的检测,具有特异性强、灵敏度高和重复性好的优点,有望用于临床实际样品的检测,进而实现临床上急性早幼粒细胞白血病的早期诊断及预后判断。A novel DNA electrochemical probe(locked nucleic acid,LNA) was designed and involved in constructing an electrochemical DNA biosensor for the detection of PML/RARα fusion gene in acute promyelocytic leukemia(APL).This biosensor was based on a "sandwich" detection strategy,which involved a pair of LNA probes,e.g.hairpin capture probe and reporter probe.Streptavidin-HRP was bound to biotin labeled at the end of reporter probe via streptavidin-biotin affinity binding.In the presence of hydrogen peroxide(H2O2),HRP catalyzed the oxidation of the substrate 3,3′,5,5′-tetramethylbenzidene(TMB) to offer an enzymatically amplified electrochemical current signal for the detection of target DNA.This sensor was applied in the direct quantitative detection of synthetic PML/RARα fusion gene in PBS buffer.The results indicated that the biosensor showed an excellent specificity to distinguish the complementary sequence and different mismatch sequences.A linear relationship between the amperometric signal and the target concentration was obtained in the range of 1.0×10-11-1.6×10-10 mol/L with a detection limit of 1.0×10-13 mol/L.In addition,the biosensor was used for the determination of PML/RARα fusion gene in human serum samples without dilution with high sensitivity,selectivity and good repeatability.This method would be expected to use in real sample for further solving the actural problems of early diagnosis and prognosis monitoring of APL.863计划资助项目(2008AA02Z433);福建省高校产学研科技重点项目(2010Y4003);国家自然科学基金资助项目(20805006;20975021);福建省自然科学基金资助项目(2010J05019

The effect of kaempferol on COX-2and iNOS expression and generated production in LPS-induced RAW 264.7 cells

目的探讨山萘酚对脂多糖(lIPOPOlySACCHArIdES,lPS)诱导rAW 264.7细胞COX-2及InOS表达的影响。方法四氮唑盐法(MOnOTE-TrAzOlIuM TEST,MTT)检测山奈酚对rAW264.7细胞生长增殖的影响,放射免疫测定法(rIA)检测山萘酚对PgE2和nO生成的影响,免疫印迹法(WESTErn blOTTIng)检测COX-2及InOS蛋白的表达。结果山萘酚抑制lPS诱导的rAW 264.7细胞PgE2和nO的生成,同时下调lPS诱导的rAW 264.7细胞COX-2及InOS蛋白的表达。结论山萘酚抑制2个诱导酶COX-2和InOS的表达,从而减少炎性产物PgE2和nO的生成,这可能是山萘酚抗炎的机制之一。Objective To study the effect of kaempferol on COX-2and iNOS expression in LPS-induced RAW 264.7cells.Methods The effects of kaempferol on the proliferation in RAW264.7cells were evaluated by monote-trazolium test assay(MTT);the effect o f kaempferol on the product of PGE2and NO in RAW264.7cells was measured by radioimmunoassay(RIA);the effects of kaempferol on the expressions of COX-2and iNOS protein in RAW264.7cells were analyzed by Western blot.Results Kaempferol inhibited the LPS-induced PGE2and NO synthesis in RAW 264.7cells.The protein expression of COX-2and iNOS in RAW264.7cells were also decreased by kaempferol.Conclusions The results show that kaempferol reduces the augmented biosynthesis of PGE2and NO by inhibiting the protein expression of COX-2and iNOS in RAW264.7cells.The finding may partly explain the anti-inflammatory effect of kaempferol.广东省社会发展领域科技计划项目(53025); 广东省重点扶持学科项目(GX0307

RAPD Analysis of the Clustering Variation of Agarucus bisporus and Cloning of the Different DNA Fragment

通过双孢蘑菇正常菌株及其丛生变异菌株基因组DNA的RAPD研究,找到一个与双孢蘑菇丛生变异可能相关的DNA片段,将其克隆并测序.GenBank查询结果表明该片段可能的部分开放读框(ORF)与多种生物的二氢硫辛酰胺脱氢酶(DLDH)的部分氨基酸序列有较高的同源性,由此提出了一个双孢蘑菇丛生变异的分子机理假设.A DNA fragment possibly related to the clustering variation of A.bisporus was found by RAPD of the genomic DNA of healthy strains and their clustering variation ones, and it was cloned and sequenced. The results of GenBank searching suggested that the possible partial ORF of the fragment had a high homology with part of amino acids of dihydrolipoamide dehydrogenase (DLDH) from several organisms, so a molecular mechanism model for the clustering variation of A.bisporus was supposed.福建省自然科学基金(B0110032)资

MOLECULAR GENETIC STUDY ON THE PEDIGREE OF HYBRID STRAIN AS2796 OF AGARICUS BISPORUS

应用PCR和凝胶电泳等技术，对双孢蘑菇杂交菌株As2796及其亲本和子代作分子遗传标记跟踪分析，结果如下: 1) 总DNA的RAPD分析表明，随着遗传代数的增加，杂种子代和出发异核体亲本间的遗传差异逐渐增大; 2) mtDNA的酶切图谱表明，亲本8213及其杂交子代具有相同的基因型，表明双孢蘑菇的mtDNA呈单亲遗传; 3) Est同工酶的PAGE图谱表明， 结合了亲本02高产特征和8213优质特征的杂交子代具有两个亲本的标记带型，证明Est同工酶标记是双孢蘑菇新菌株特性预测或鉴定的有效指标。The pedigree of the hybrid A.bisporus strain As2796 was analyzed through molecular genetic markers tracking. The results were as follows: 1)RAPD analysis of total DNA showed that with the addition of the number of genetic generations， the genetic differences between hybrid offspring and their original heterokaryotic parents increased. 2)The patterns of mtDNA digested by endonucleases showed that the hybrid offspring had the same mitochondrial genotype as the parent 8213， indicating that the mtDNA of A.bisporus was inherited from one of the parents. 3)PAGE patterns of esterase isozyme(Est) showed that the hybrid offsprings which combined both the characteristic of high yield of parent 02 and good quality of 8213 had the same marked bands of their parents. The Est isozyme marker was found to be an effective index of new strains' characteristic prediction or determination of A.bisporus.福建省自然科学基金重点资助项目!（C9820005

Analysis of the Protein Expression of Agaricus bisporus Strains after Growing at High Temperature

对 2 4℃常温培养和 32℃诱导培养的双孢蘑菇 82 13、0 2、2 796、4 60 7菌株的菌丝体总蛋白质的 SDS- PAGE分析比较 ,发现 2 796和 4 60 7菌株在 4 3k D～ 110 k D之间有明显的差异蛋白质出现 ,而 0 2和 82 13菌株的蛋白质差异带不明显 .这些高温诱导特异表达的蛋白质可能与双孢蘑菇的热耐受相关 .Agaricus bisporus strains (02821327964607) from a genetic family were cultured at different temperature (32 ℃ and 24 ℃). Analysis of the protein pattern with SDS-PAGE of those strains showed that strain 2796 and 4607 have many induced protein bands between 43 kD～110 kD, but strain 02 and strain 8213 have few induced protein bands. Those results suggested that the thermotolerance character is related to the different genomic construct of different strains.福建省自然科学基金资助 (C982 0 0 0 5